Multiple Mechanisms of Unfolded Protein Response-Induced Cell Death

The American Journal of Pathology, Vol. 185, No. 7, July 2015

ajp.amjpathol.org ASIP COTRAN EARLY CAREER INVESTIGATOR AWARD LECTURE Multiple Mechanisms of Unfolded Protein ResponseeInduced Cell Death

Nobuhiko Hiramatsu, Wei-Chieh Chiang, Timothy D. Kurt, Christina J. Sigurdson, and Jonathan H. Lin

From the Department of Pathology, University of California-San Diego, La Jolla, California

Accepted for publication March 26, 2015. Eukaryotic cells fold and assemble membrane and secreted proteins in the endoplasmic reticulum (ER), before delivery to other cellular compartments or the extracellular environment. Correctly folded pro- Address correspondence to Jonathan H. Lin, Department of teins are released from the ER, and poorly folded proteins are retained until they achieve stable Pathology, University of conformations; irreparably misfolded proteins are targeted for degradation. Diverse pathological in- California-San Diego, 9500 sults, such as amino acid mutations, hypoxia, or infection, can overwhelm ER protein quality control, Gilman Dr, La Jolla, CA leading to misfolded protein buildup, causing ER stress. To cope with ER stress, eukaryotic cells activate 92093-0612. E-mail: jlin@ the unfolded protein response (UPR) by increasing levels of ER protein-folding enzymes and chaper- ucsd.edu. ones, enhancing the degradation of misfolded proteins, and reducing protein translation. In mammalian cells, three ER transmembrane proteins, inositol-requiring enzyme-1 (IRE1; official name ERN1), PKR-like ER kinase (PERK; official name EIF2AK3), and activating transcription factor-6, control the UPR. The UPR signaling triggers a set of prodeath programs when the cells fail to successfully adapt to ER stress or restore homeostasis. ER stress and UPR signaling are implicated in the pathogenesis of diverse diseases, including neurodegeneration, cancer, diabetes, and inflammation. This review dis- cusses the current understanding in both adaptive and apoptotic responses as well as the molecular mechanisms instigating apoptosis via IRE1 and PERK signaling. We also examine how IRE1 and PERK signaling may be differentially used during neurodegeneration arising in retinitis pigmentosa and prion infection. (Am J Pathol 2015, 185: 1800e1808; http://dx.doi.org/10.1016/j.ajpath.2015.03.009)

The endoplasmic reticulum (ER) is an essential organelle domains.1 In response to ER stress, IRE1 undergoes oligomeric responsible for folding of secreted and membrane proteins assembly, transautophosphorylation by its kinase domain, and and lipid and sterol biosynthesis, and it is a major site of activation of its distal RNase activity.2,3 In metazoans, the free calcium storage within the cell. Cells have evolved a RNase activity of activated IRE1 and the RtcB tRNA ligase unique homeostatic mechanism, termed the unfolded pro- splice out a small intron from the X-box binding protein-1 tein response (UPR), to ensure that the ER can adapt to (Xbp1) mRNA to produce XBP1s transcription factor.4e8 changing environmental and physiological demands of its functions. In mammalian cells, the UPR is controlled by Supported by NIH grants R01EY020846, R01NS069566, R01NS076896, and R01NS088485 and VA grant BX002284. the ER resident transmembrane proteins, inositol-requiring Disclosures: None declared. enzyme-1 (IRE1; ofﬁcial name ERN1), PKR-like ER The American Society for Investigative Pathology Cotran Early Career kinase (PERK; ofﬁcial name EIF2AK3), and activating Investigator Award recognizes early career investigators with demonstrated transcription factor-6 (ATF6). excellence as an investigator with recently established or emerging indepen- IRE1 is a transmembrane protein that controls a UPR signal dence and with a research focus leading to an improved understanding of the conceptual basis of disease. Jonathan H. Lin, recipient of the ASIP 2013 Cotran transduction pathway conserved from yeast to mammals 1 Early Career Investigator Award, delivered a lecture entitled Endoplasmic Re- (Figure 1). IRE1 bears a luminal domain coupled across the ER ticulum Stress in Disease Pathogenesis on April 22, 2013, at the annual meeting membrane to cytosolic kinase and endoribonuclease (RNase) of the American Society for Investigative Pathology in Boston, MA.

Normal Stress Unfolded Proteins

BiP IRE1 PERK ATF6

SH S SH S

P P b b P P K K Z P K P Z i K K i

p R P P P P p R P P

P eIF2α eIF2α S1P S2P

XBP1s 40S bZip 60S

XBP1s bZip

Figure 1 Endoplasmic reticulum (ER) stress activates inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor-6 (ATF6) intracellular signal transduction pathways of the unfolded protein response (UPR). In normal condition, the UPR transducers, IRE1, PERK, and ATF6, associate with BiP to prevent UPR. On accumulation of misfolded proteins in the ER lumen, BiP dissociates to activate UPR transducers. IRE1 bears a luminal domain coupled across the ER membrane to cytosolic kinase (K) and endoribonuclease (RNase) domains (R). In response to ER stress, IRE1 undergoes oligomeric assembly, transautophosphorylation by its kinase domain, activating its distal RNase activity. Activated IRE1 splices out small intron from the X-box binding protein-1 (Xbp1) mRNA to generate active transcription factor XBP1s. The PERK protein bears luminal domain coupled across the ER membrane to K. In response to ER stress, PERK dimerizes and subsequently activates its cytosolic kinase domain. PERK’s kinase recognizes and phosphorylates eukaryotic translation initiation factor 2 subunit alpha (eIF2a), leading to attenuation of global protein translation. ATF6 bears an ER-tethered transcription factor. In response to ER stress, ATF6 migrates from the ER to the Golgi apparatus, where site 1 and 2 proteases (S1P and S2P, respectively) cleave its luminal and transmembrane domains, and release the cytosolic portion of ATF6 containing the bZIP transcriptional activator domain. Cleaved ATF6 fragment translocates to the nucleus to serve as a transcription factor.

The transcriptional targets of XBP1 are highly enriched portion of ATF6 contains the bZIP transcriptional activator for ER-associated protein degradation (ERAD) factors, ER domain, and after cleavage, this ATF6 fragment migrates to chaperones, and enzymes required for lipid biosynthesis the nucleus to transcriptionally up-regulate ER chaperones and protein glycosylation across diverse mammalian cell and ERAD components, thereby enhancing ER protein- types.9e14 Up-regulation of these molecules by IRE1-to- folding capacity and efficiency of ERAD.10,12,18,20 Inter- XBP1s induction therefore enhances the ER’s capacity to estingly, ATF6 also transcriptionally up-regulates Xbp1, better fold new proteins as well as target irreparably thereby facilitating IRE1 signal transduction by increasing damaged proteins for retrotranslocation out of the ER for levels of IRE1’s RNase substrate, Xbp1 mRNA.21 degradation by proteasomes in the cytosol (Figure 1). Put together, these initial transcriptional and translational The ER transmembrane protein PERK regulates another effects of IRE1, PERK, and ATF6 signaling help cells adapt UPR signaling pathway in metazoans15 (Figure 1). On ER to ER stress by enhancing the fidelity of protein folding, stress, PERK oligomerizes and activates its cytosolic kinase increasing the degradation of damaged/misfolded proteins, domain.15 PERK’s kinase phosphorylates eukaryotic trans- and suppressing new protein synthesis. However, if these lation initiation factor 2 subunit alpha (eIF2a)onSer51, actions fail to restore ER homeostasis and ER stress persists, inhibiting the guanine nucleotide exchange factor eIF2B, which UPR signaling consequently triggers maladaptive proapo- converts inactive GDP-bound eIF2 to its active GTP form.15,16 ptotic programs, many of which are specifically activated Active eIF2 complex is needed to form the GTP-tRNAMet through the IRE1 and PERK pathways. ternary complex required for translation initiation. Therefore, eIF2a phosphorylation leads to translation inhibition that helps alleviate ER stress by reducing the load of new polypeptides IRE1 Signaling through RIDD, c-Jun N-Terminal that require assembly and folding in the ER compartment.17 Kinase, and BCL2 ATF6 bears an ER-tethered bZip transcription factor that regulates a third UPR signal transduction pathway18 Seminal mechanistic studies from the laboratory of Walter (Figure 1). In response to ER stress, ATF6 migrates from and colleagues have revealed that IRE1 undergoes dynamic the ER to the Golgi apparatus, where site 1 protease and site conformational and functional changes as a function of the 2 protease cleave its luminal and transmembrane domains to duration of ER stress. In response to acute ER stress, IRE1 release the cytosolic portion of ATF6.18,19 The cytosolic quickly forms oligomeric clusters in the ER plane, but IRE1

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Normal Acute Phase Chronic Phase

P P P ? ? P K P K P P K K P P P P P R R R R TRAF2 ASK1 XBP1s RIDS RIDD P DR5 JNK BiP Cyp1a2 & Cyp2e1 ERAD Ces1 & Angptl3 Chaperone IRE1α Lipid Biosynthesis miRNAs Glycosylation

Figure 2 Consequences of acute and chronic inositol-requiring enzyme 1 (IRE1) activation. In response to acute endoplasmic reticulum (ER) stress, IRE1 undergoes oligomeric assembly, undergoes transautophosphorylation by its kinase domain, and activates its distal RNase activity. Activated IRE1’s RNase splices out a small intron from the X-box binding protein-1 (Xbp1) mRNA to produce the active transcription factor XBP1s. IRE1-to-XBP1s induction enhances the ER’s capacity by up-regulation of gene sets involved in ER-associated protein degradation (ERAD), ER chaperones, lipid biosynthesis, and protein glycosylation. In the chronic phase of IRE1 activation, IRE1’s RNase domain cleaves ER-targeted mRNAs in a phenomenon termed regulated IRE1-dependent mRNA decay (RIDD). Most RIDD-targeted mRNAs are disposed. In contrast, IRE1-dependent cleavage of the 30 untranslated region of BiP mRNA in Schizo- saccharomyces pombe stabilizes BiP mRNA, thereby increasing BiP protein levels to cope with ER stress. There is likely a regulated IRE1-dependent mRNA stabilization (RIDS) rather than RIDD, which may be a new mode by which IRE1’s RNase positively regulates mRNAs. The TRAF2 adaptor protein binds to IRE1 and the MAPKKK ASK1 to activate downstream molecules such as c-Jun N-terminal kinase (JNK), p38, and extracellular signaleregulated kinase (ERK). However, it is unclear which phase of IRE1 activity can interact with TRAF2-ASK1. DR5, death receptor 5. subsequently dissociates if ER stress persists (Figure 2).22,23 Recently, IRE1’s RNase was found to cleave the 30 un- Interestingly, Xbp1 mRNA splicing only occurs during the translated region of BiP mRNA in Schizosaccharomyces acute phase.23e25 During the chronic phase of ER stress, pombe, but this truncation surprisingly stabilized, rather than IRE1’s RNase substrate specificity is altered to cleave pri- promoted, the decay of the remaining BiP mRNA, thereby marily ER-targeted mRNAs in a process termed regulated increasing BiP protein levels during ER stress.35 Regulated IRE1-dependent mRNA decay (RIDD)26e28 (Figure 2). IRE1-dependent mRNA stabilization, rather than RIDD, may In contrast to Xbp1 mRNA, RIDD targets are not ligated be a new mode by which IRE1’s RNase positively regulates after IRE1 cleavage, and most mRNA fragments cleaved mRNAs (Figure 2). Recent biochemical studies have sug- through RIDD are degraded. RIDD’s physiological signifi- gested that IRE1 uses RIDD at intense ER stress levels, cance varies widely and can confer protective or proapoptotic whereas Xbp1 mRNA splicing is initiated at much lower effects, depending on the cellular function of the mRNA being levels of ER stress25 (Figure 2). IRE1’s decision to trigger targeted. RIDD-mediated cleavage of death receptor 5 (DR5) RIDD, regulated IRE1-dependent mRNA stabilization, or mRNA enhances cell survival during ER stress by reducing Xbp-1 mRNA splicing may be dependent on the intensity production of proapoptotic DR5 protein.29 RIDD-mediated of the stress and the ability of various cell types to respond to cleavage of cytochrome P450 enzyme mRNAs in liver con- that stress. fers resistance to liver damage after acetaminophen overdose The IRE1 oligomeric clusters formed by ER stress also act by preventing P450-mediated generation of hepatotoxic by- as molecular scaffolds to recruit other proteins and nucleate products.30 The mRNA fragments produced by RIDD cleav- the formation of stress signal transduction sites at the ER lipid age can trigger inflammation by engaging with the cytosolic bilayer22,23 (Figure 2). For example, TRAF2 adaptor protein RIG1 RNA virus innate immunity sensor.31 RIDD-mediated binds to IRE1 as well as the ASK1 MAPKKK to activate loss of lipid metabolism mRNAs can alter plasma lipid cytosolic signaling kinases, such as C-Jun N-terminal kinase, levels in mice.32 Ire1 mRNA itself is a RIDD substrate, and p38, and extracellular signaleregulated kinase during ER RIDD may act as an autoregulatory brake on IRE1 signaling stress36,37 (Figure 2). IRE1 also interacts with the RACK1 by down-regulating Ire1 mRNA levels.33 miRNA precursors adaptor protein to recruit phosphatases to the ER membrane have also been identified as RIDD substrates in vitro.34 during ER stress.38 IRE1 forms protein-protein interactions Disruption of miRNA maturation by RIDD cleavage may with BCL2 family proteins, such as BAX and BAK, and further affect multiple biological processes by modulating the BI-1 BCL2 regulatory protein.39,40 IRE1 also binds miRNA-mRNA interactions throughout the cell. cytoskeletal nonmuscle myosin II during ER stress.41

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Normal Acute Phase Chronic Phase

P K K P P P K P P P K KP P P P P

P eIF2α eIF2α P eIF2α Translation Attenuation 40S 60S GADD34 ATF4 Degradation IAPs Casp Translation Attenuation Inactive

Redox CHOP Golgi IAPs Genes

DR5 Casp Cell Death Active

Figure 3 Consequences of acute and chronic PKR-like endoplasmic reticulum (ER) kinase (PERK) activation. PERK has a kinase domain (K), and phosphorylates eukaryotic translation initiation factor 2 subunit alpha (eIF2a). In the acute phase, PERK-eIF2aP attenuates overloading the proteins into ER. On chronic activation of PERK signaling, expression of activating transcription factor-4 (ATF4) is transnationally up-regulated, which regulates cell fate. GADD34 dephosphorylates eIF2aP to eIF2a, and protein translation is reinitiated. Expression of ATF4 causes oxidative stress. Proapoptotic transcription factor CHOP is transcriptionally induced by ATF4, and its translation is also enhanced by ATF4. Death receptor 5 (DR5; ofﬁcial name TNFRSF10B) is a CHOP target gene, and abundant DR5 protein forms oligomer at the Golgi apparatus, which activates caspase (Casp)8 without requirement of any ligand. Inhibitors of apoptosis proteins (IAPs) are key cell death regulators in metazoans, through their suppression of caspases. Recent studies link PERK-eIF2aP-ATF4 signaling to IAP regulation during ER stress. In response to chronic ER stress, IAP levels decrease speciﬁcally through the actions of PERK, but not IRE1 or ATF6 branches of the unfolded protein response. The eIF2aP attenuates de novo IAP synthesis, particularly X-linked IAP, and ATF4’s transcriptional activity destabilizes extant XIAP protein.

These IRE1-centered protein-protein interactions can in- fibroblasts and neurons also evokes oxidative stress and influence the sensitivity of IRE1 activation in response creases cell death.45,47 These findings point to ATF4 as an to ER stress. IRE1-centered protein-protein interactions important determinant in regulating cell fate during ER stress, could also act conversely to influence cellular signaling with too little and too much ATF4 both producing deleterious processes and structures in other cellular compartments effects. during ER stress. One mechanism by which ATF4 can promote cell death is via transcriptional up-regulation of the GADD34 phosphatase (Figure 3). GADD34 dephosphorylates eIF2aPto Proapoptotic Consequences of PERK Signaling eIF2a.48 Dephosphorylation of eIF2aP removes the translational brake initially generated by PERK activation and PERK signaling down-regulates translation from most leads to more protein synthesis and thereby protein folding mRNAs, thereby restricting de novo peptide loading onto the demands on the ER. Indeed, Han et al45 found that ATF4 ER (Figure 1). This cytoprotective event by PERK to eIF2aP overexpression increased protein synthesis concomitant to occurs rapidly (Figure 3). Rare mRNAs bearing 50 upstream increasing cell death. Furthermore, chemical inhibition of open reading frames, including the mRNAs encoding the GADD34’s dephosphorylation of eIF2aP by the salubrinal ATF4, ATF5, and CHOP transcriptional activators, are para- and guanabenz compounds protects cells from ER doxically translated more efficiently during the phosphorylated stresseinduced cell death.49e51 state of eIF2a (Figure 3).42e44 ATF4’s transcriptional targets A second mechanism by which ATF4 promotes cell death include genes involved in amino acid metabolism, oxidore- is via transcription of the proapoptotic Chop gene (official ductases required for disulfide bond formation in the ER, name DDIT3), whose translation is also enhanced by eIF2aP several ubiquitin ligases, GADD34 phosphatase, and CHOP (Figure 3).16,43,44,52,53 By dual transcriptional and trans- transcription factor.45e47 ATF4-null mouse embryonic fibro- lational up-regulation, CHOP is highly enriched when PERK blasts are sensitive to oxidative stress and require supplemental is strongly activated. CHOP’s role as a proapoptotic tran- reducing compounds for survival and growth in cell culture.46 scription factor has been clearly shown in vitro where CHOP- Interestingly, ATF4 overexpression in mouse embryonic null cells are resistant to cell death induced by the chemical

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ER toxins, tunicamycin, and thapsigargin.53 Several of diseases associated with ER stress, retinitis pigmentosa and CHOP’s transcriptional targets are implicated in apoptosis, prion infection. including the apoptotic Bim (official name BCL2L11) 54,55 and Puma (official name BBC3)Bcl-2familygenes, Divergent Mechanisms of ER StresseInduced fi fi Trb3 (of cial name TAS2R13), and Dr5 (of cial name Neurodegeneration TNFRSF10B).29,56,57 DR5 can signal cell death by activating caspase-8.29,57 During ER stresseinduced cell death, DR5 ER-Associated Degradation in Retinitis Pigmentosa protein accumulates in Golgi apparatus, where it oligomerizes, leading to activation of cytosolic caspase 8. Interestingly, DR5 Retinitis pigmentosa is a human blinding disease arising mRNA is also down-regulated by IRE1-mediated RIDD, and from photoreceptor cell death in the eye. Photoreceptors are the balance between the opposing effects of IRE1 and PERK on specialized sensory neurons that detect light and activate DR5 levels may tip whether UPR selects cell survival or cell retinal circuitry to transmit visual information to the brain. death during ER stress.29 Photoreceptors accomplish this feat using the visual Some types of ER stresseinduced damage and cell pigment, rhodopsin, a G-proteinecoupled transmembrane death are unlikely to be mediated via CHOP induction. receptor protein covalently linked to 11-cis-retinal.64 Comprehensive RNA-sequencing and microarray studies Rhodopsin is essential for photoreceptor function and sur- saw minimal transcriptional induction of previously vival, and rhodopsin knockout mice (Rho/) develop early identified apoptotic genes after forced Chop expres- retinal degeneration.65,66 More than 100 rhodopsin muta- sion.45 Furthermore, forced CHOP expression itself does tions have been identified in families with heritable types of not trigger cell death in vitro,45,58 indicating that other retinitis pigmentosa.67 Many of these mutations generate proapoptotic hits are necessary for ER stresseinduced misfolded rhodopsin proteins that are aggregation prone and cell death. retained in the ER.24,68e71 Recent studies in mouse models Inhibitors of apoptosis proteins (IAPs) are key cell death of retinitis pigmentosa have shed light into how the UPR in regulators in metazoan organisms through their suppression photoreceptors copes with mutant rhodopsins and influences of caspases.59 Recent studies link PERK-eIF2aP-ATF4 the disease process. signaling to IAP regulation during ER stress. In response to The P23H rhodopsin mutation is the most common cause of chronic ER stress, IAP levels decrease significantly in many heritable retinitis pigmentosa in North America, and photore- mammalian cell types, specifically through the actions of the ceptors carrying the mutation generate misfolded rhodopsin e PERK, but not IRE1 or ATF6, branches of the UPR.60 63 proteins that do not traffic normally to the rod photoreceptor PERK signaling attenuates de novo X-linked IAP (XIAP) outer segment. A P23H rhodopsin knock-in mouse closely protein synthesis via eIF2aP and also promotes extant XIAP mirrors the spatial distribution and temporal progression of protein degradation by ATF4 transcriptional activity photoreceptor cell death and vision loss found in patients with (Figure 3). In contrast, CHOP had no effect on XIAP levels. this mutation.72 Analysis of these mice indicates that these Loss of XIAP enhances sensitivity to ER stresseinduced photoreceptors use an unusual, customized UPR tailored to cell death, and overexpression of XIAP protects cells from cope with P23H rhodopsin.73 IRE1’s induction of XBP1s, and ER stress, and interestingly, synergizes with the absence of the transcriptional up-regulation of ERAD by XBP1s, was CHOP to induce even greater resistance to ER stresse seen in photoreceptors expressing P23H rhodopsin.73 induced cell death than Chop / alone.58 These findings Concomitant with ERAD up-regulation, P23H rhodopsin show that PERK-eIF2aP-ATF4 signaling promotes multiple protein is found to be robustly ubiquitinated and almost proapoptotic hits within the cell, including the induction of entirely degraded in these photoreceptors73 (Figure 4A). In CHOP and suppression of IAPs (Figure 3). These effects of contrast, other IRE1-mediated signaling events, including chronic PERK signaling, therefore, generate a cellular c-Jun N-terminal kinase activation or RIDD, are not observed milieu conducive for efficient caspase activation by removal in these photoreceptors.73 Furthermore, minimal activation of of caspase inhibitors. the PERK signaling pathway is seen, with no changes in The ability of IRE1 and PERK signaling to activate ATF4, CHOP, or IAP levels in P23H rhodopsin-expressing multiple distinct proapoptotic circuits provides attractive photoreceptors.73 These findings reveal that the dominant ef- mechanisms to link ER stress to disease pathogenesis and fect of UPR in photoreceptors of P23H rhodopsin knock-in progression. Physiological ER stresses vary tremendously mice is the induction of ERAD to degrade and clear mutant with respect to their intensity and their cause (eg, hypoxia rhodopsin, which is accomplished through a preferential use of versus genetic mutation). Important questions for defining the parts of the UPR regulating ERAD, such as IRE1’sin- the role of UPR as disease mechanism include the duction of XBP1. PERK’s proapoptotic functions through following: Which UPR signaling events are activated by a modulation of ATF4, CHOP, and IAPs are not used in these physiological ER stress? What is the consequence of UPR photoreceptors. Consistently, Chop/ fails to delay retinal activation in the cellular and tissue context of a specific degeneration in P23H rhodopsin knock-in photoreceptors, and disease? In the subsequent sections, we examine how UPR in other genetically modified mouse lines expressing other activation and function contribute to the pathogenesis of two rhodopsin mutations seen in retinitis pigmentosa.73e75

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Figure 4 A: Rhodopsin (Rho) is robustly degraded during retinal degeneration. Light micrographs of wild-type and P23H knock-in mouse retinas at postnatal (P) day 15. At P15, both rod outer segments (OSs) and rod inner segments (ISs) are shorter in RhoP23H/þ mice compared with those of the Rhoþ/þ mice, and significantly shortened in RhoP23H/P23H mice. The outer nuclear layer (ONL) is also significantly thinner in RhoP23H/P23H mice. Rhodopsin protein levels are significantly diminished in RhoP23H/P23H mice. Retinal protein lysates were collected from Rhoþ/þ, RhoP23H/þ, and RhoP23H/P23H mice at P15. Rhodopsin is detected by immunoblotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. B: High prion protein (PrP) levels are maintained during prion infection. Hematoxylin and eosinestained hippocampal sections from mock- or prion-inoculated transgenic mice expressing human PrP reveal neuronal necrosis (arrowhead) and spongiform degeneration only in prion-infected mice. Total PrP levels (PrPC þ PrPSc) in brain are similar in mock- and prion-infected mice by immunoblotting (20 mg protein per well). Samples treated with 100 mg/mL proteinase K (PK) reveal PK-resistant PrPSc only in prion- infected brain (100 mg protein per well). Actin served as a loading control (the actin bands are above the PrP in the undigested lanes). This blot was developed using monoclonal antibodies 3F4 against PrP (Millipore, Billerica, MA) and GT5412 against actin (Genetex, Irvine, CA). PrP contains two potential n-glycosylation sites and, thus, migrates as three bands corresponding to diglycosylated, monoglycosylated, or unglycosylated PrP. Scale bars: 10 mm(A); 50 mm(B).

What drives photoreceptor cell death if PERK’spro- and UPR activation have also been observed in prion infec- apoptotic signals are not activated? Rhodopsin is essential tion in vivo83e85 as well as transgenic mice expressing mutant for photoreceptor function, structure, development, and PrPC.86 survival, and Rho/ mice develop early retinal degen- Genetic and chemical modulation of different UPR eration.65,66 In P23H rhodopsin knock-in animals, pathways in prion-infected models has revealed intriguing rhodopsin protein degradation occurs as soon as photo- and surprising differences from mutant rhodopsin models in receptors are born, and loss of rhodopsin precedes any the role of UPR signaling pathways in cellular degeneration. photoreceptor cell death (Figure 4A).73 Disruption of PrPC-to-PrPSc conversion and neurodegeneration are un- rhodopsin protein homeostasis by ERAD is likely to be a changed in mice deﬁcient in neuronal Xbp1/ compared key trigger for photoreceptor cell death. with controls.85 In contrast, genetic or chemical inhibition of PERK pathway signaling using GADD34 overexpression or PERK Signaling in Prion Diseases the PERK inhibitor glycogen synthetase kinase 2606414 ameliorates neurodegeneration in prion-infected mice, whereas Prion diseases are fatal neurodegenerative disorders arising activating the PERK pathway using salubrinal worsens prion- from conversion of the normal cellular prion protein, PrPC, associated neurotoxicity.83,84 These studies reveal a direct role into a misfolded and self-templating conformer, PrPSc.PrPC for the PERK pathway in prion disease pathogenesis, and is highly conserved among mammals and ubiquitously suggest that IRE1 signaling, at least through XBP1s genera- expressed, yet the functions attributed to PrPC are diverse and tion, is dispensable in this process. include maintenance of myelin, nomal synaptic function, and How is the PERK signaling critical in the pathogenesis of e neuroprotection.76 79 PrPC is a glycosylphosphatidylinositol- prion disease, yet unnecessary for mutant rhodopsin- anchored glycoprotein that undergoes folding and post- induced cell death? One fundamental difference between translational modiﬁcations within the ER and secretory these diseases is that prion conversion likely occurs on the pathway. In cell culture, PrPSc replication triggers ER stress, cell membrane or in an endolysosomal compartment; thus, resulting in the aberrant accumulation of PrP in the cytosol PrPSc escapes the misfolded protein clearance and degra- and further enhancing the formation of PrPSc.80e82 ER stress dation mechanisms triggered during UPR activation86

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(Figure 4B). Indeed, levels of the PrPSc isoform increase tRNA ligase complex mediates splicing of XBP1 mRNA and controls substantially by the terminal stage of disease (Figure 4B). antibody secretion in plasma cells. EMBO J 2014, 33:2922e2936 The accumulation of PrP in the brain likely causes chronic 8. Kosmaczewski SG, Edwards TJ, Han SM, Eckwahl MJ, Meyer BI, Peach S, Hesselberth JR, Wolin SL, Hammarlund M: The RtcB RNA ER stress, leading to strong PERK signaling and its subse- ligase is an essential component of the metazoan unfolded protein quent proapoptotic signaling cascade (Figure 2). In contrast, response. EMBO Rep 2014, 15:1278e1285 misfolded rhodopsin is degraded so quickly and efficiently 9. Lee AH, Iwakoshi NN, Glimcher LH: XBP-1 regulates a subset of (Figure 4A) that the strength and duration of ER stress do endoplasmic reticulum resident chaperone genes in the unfolded e not increase to a threshold necessary for strong PERK protein response. Mol Cell Biol 2003, 23:7448 7459 10. Shoulders MD, Ryno LM, Genereux JC, Moresco JJ, Tu PG, Wu C, activation. Thus, prion infection may represent a class of ER Yates JR 3rd, Su AI, Kelly JW, Wiseman RL: Stress-independent stresseassociated diseases in which PERK’s proapoptotic activation of XBP1s and/or ATF6 reveals three functionally diverse signaling output prevails when UPR protein quality control ER proteostasis environments. Cell Rep 2013, 3:1279e1292 mechanisms fail to remove the misfolded proteins. Mis- 11. Yamamoto K, Yoshida H, Kokame K, Kaufman RJ, Mori K: Dif- folded rhodopsins may represent another class of ER ferential contributions of ATF6 and XBP1 to the activation of e endoplasmic reticulum stress-responsive cis-acting elements ERSE, stress associated diseases in which UPR protein quality UPRE and ERSE-II. J Biochem 2004, 136:343e350 control mechanisms remove the misfolded proteins but, in 12. Yamamoto K, Sato T, Matsui T, Sato M, Okada T, Yoshida H, doing so, disrupt vital cellular structures or processes Harada A, Mori K: Transcriptional induction of mammalian ER required for cell viability. quality control proteins is mediated by single or combined action of ATF6alpha and XBP1. Dev Cell 2007, 13:365e376 13. Shaffer AL, Shapiro-Shelef M, Iwakoshi NN, Lee AH, Qian SB, Tailoring the UPR to Fit a Specific Disease Zhao H, Yu X, Yang L, Tan BK, Rosenwald A, Hurt EM, Petroulakis E, Sonenberg N, Yewdell JW, Calame K, Glimcher LH, The UPR signaling pathways are found in all cell types and Staudt LM: XBP1, downstream of Blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in activate broad transcriptional, translational, and post- plasma cell differentiation. Immunity 2004, 21:81e93 translational programs to help cells cope with ER stress. 14. 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